The immunoassay is a well established technique for detecting targets in a biological sample (e.g. blood or urine) by employing an antibody specific to that target. Example targets may include cardiac markers such as troponin used to indicate the occurrence of a heart attack, or C-Reactive protein which is an indicator of infection. A common format is the “enzyme-linked immunosorbent assay” or “sandwich ELISA” assay, which requires such antibodies to be bound to a surface such as, for example, the wall of the reaction device or vessel. The use of polymer-coated beads as such a surface is known (e.g. Decker, GB2016687, published Sep. 26, 1979).
FIG. 1 illustrates the process of a typical immunoassay. FIG. 1 in particular illustrates a sequence of combination of droplets of sample and reagent to carry out such an assay. FIG. 1a shows a first of droplets 2 containing beads 4 with primary antibody 6 bound to it. The second of droplets 2 contains the target 8. When the first and second droplets are mixed, the target binds to the bead-antibody complex 10. In a next step (FIG. 1 b) a further third droplet 2 is introduced containing a secondary antibody 12 conjugated to a fluorescent component. This then binds to those targets that were already bound to the first antibody forming a complex of bead, primary antibody, target and secondary antibody 14. FIG. 1c illustrates the key step in the assay, known as washing. The purpose of washing is to remove the unbound secondary antibody 12, which would give a false positive signal, leaving only the bound antibody complex 14. As such, this step is critical in ensuring the accuracy of the assay. The droplet is mixed with a wash buffer 16. The beads are then separated from the unbound antibody by suitable means, leaving only bound secondary antibodies 14. When light of a suitable wavelength 18 is incident on the secondary antibody, it fluoresces and emits light at a longer wavelength 20 that may be detected (FIG. 1d). The intensity of such light is proportional to the concentration of bound secondary antibody, and hence to the concentration of original target.
Microfluidics is a rapidly expanding field concerned with the manipulation and precise control of fluids on a small scale, often dealing with sub-microliter volumes. There is growing interest in its application to chemical or biochemical assay and synthesis, both in research and production, and applied to healthcare diagnostics (“lab-on-a-chip”). In the latter case, the small nature of such devices allows rapid testing at the point of need using much smaller clinical sample volumes than for traditional lab-based testing.
Electrowetting on dielectric (EWOD) is a well-known technique for manipulating discrete droplets of fluid by application of an electric field. It is thus a candidate technology for microfluidics for lab-on-a-chip technology. An introduction to the basic principles of the technology can be found in “Digital microfluidics: is a true lab-on-a-chip possible?”, (R. B. Fair, Micofluid Nanofluid (2007) 3:245-281).
A common means of carrying out the separation illustrated in FIG. 1c is to employ beads that are paramagnetic or ferromagnetic, for example by having a ferrite core. In this case the beads may be immobilized in the presence of a magnetic field. This may be provided, for example, by an electromagnet or a permanent magnet (e.g. neodymium). The beads move in the direction of the magnetic field gradient and hence magnets shaped to enhance magnetic field density and gradient may be advantageous. Once immobilized the droplet containing the unbound antibody may be moved away from the beads. Conversely, the droplet may be held still whilst the magnet, and hence beads, are moved. This process is illustrated in FIG. 2. In FIG. 2a the bound antibody complexes 14 and unbound antibodies 12 are combined with buffer 16 in the presence of magnet 22. They are separated to leave bound antibody only in the original droplet (FIG. 2b). The droplet may be controlled by various means, for example by an EWOD system. However, such an approach tends to only result in a low dilution factor of the unbound antibody due to the simple mixing of antibody droplet and buffer, which constitutes inefficient washing. Therefore, the process of FIG. 2 requires many repeats of the washing cycle to achieve sufficient dilution, which increases assay time and reagent usage. A system using magnets and EWOD is disclosed in Pamula, et al., US2007/0241068, published on Oct. 18, 2007. Pamula et al, however, does not describe how to achieve high efficiency washing.
Beebe et al. (“One step purification of nucleic acid for gene expression and analysis via Immiscible Filtration Assisted by Surface Tension”, Beebe et al, Lab Chip 2011,11,1747 (2011)) discloses bead-based washing in a fixed chamber format but do not describe any form of droplet control.
Campbell et al., US20120034684A1, published on Feb. 9, 2012, discloses the use of bead-based immunoassay in a disposable cartridge format, but does not describe details on the washing method.